Isolation of factors that associate directly or indirectly with chromatin

Abstract

Methods for isolating non-coding nucleic acids that are associated with chromatin at a target genomic locus are provided. The methods comprise the steps of obtaining a sample that comprises a target genomic DNA sequence and one or more non-coding nucleic acids associated with that DNA sequence; contacting the sample with at least one oligonucleotide probe that comprises a sequence that is complimentary to and capable of hybridising with at least a portion of the target DNA sequence, wherein the oligonucleotide probe comprises at least one modified nucleotide analogue and wherein the oligonucleotide probe further comprises at least one affinity label; allowing the at least one oligonucleotide probe and the target DNA sequence to hybridise with each other so as to form a probe-target hybrid; isolating the probe-target hybrid from the sample by immobilizing the probe-target hybrid through a molecule that binds to the at least one affinity label; and eluting the one or more non-coding nucleic acids that are associated with the target genomic DNA sequence. Also provided are probes suitable for use in the methods of the invention. The methods and probes of the invention are suited to identification of non-coding RNAs including microRNAs and snoRNAs that are associated with chromatin remodelling.

1 . A method for isolating one or more non-coding nucleic acids associated with a target DNA sequence that is comprised within chromatin, comprising the steps of:
 (a) obtaining a sample that comprises a target DNA sequence as well as one or more non-coding nucleic acids that are associated with the target DNA sequence;   (b) contacting the sample with at least one oligonucleotide probe that comprises a sequence that is complimentary to and capable of hybridising with at least a portion of the target DNA sequence, wherein the oligonucleotide probe comprises at least one modified nucleotide analogue and wherein the oligonucleotide probe further comprises at least one affinity label;   (c) allowing the at least one oligonucleotide probe and the target DNA sequence to hybridise with each other so as to form a probe-target hybrid;   (d) isolating the probe-target hybrid from the sample by immobilizing the probe-target hybrid through a molecule that binds to the at least one affinity label; and   (e) eluting the one or more non-coding nucleic acids that are associated with the target DNA sequence.   
     
     
         2 . The method of  claim 1  wherein the one or more non-coding nucleic acids comprises non-coding RNA. 
     
     
         3 . The method of  claim 1  wherein the one or more non-coding nucleic acids comprises one or more micro-RNAs. 
     
     
         4 . The method of  claim 1 , wherein the at least one oligonucleotide probe comprises at least one group that conforms to general formula I, set out below:
   A—[C] n —X  I
   wherein A includes one or more affinity labels tethered to a modified nucleotide analogue X by a spacer group C of n atoms in length; A comprises a hapten or an immuno-tag; and wherein the nucleotide analogue X is selected from a peptide nucleic acid (PNA); a 2′ modified ribonucleotide analogue, including 2′-O—R sugar modifications, wherein R is selected from the group consisting of: methyl; ethyl; C 1  to C 5  alkyl; and aryl; a 2′ substituted ribonucleotide analogue, including 2′-C and 2′-deoxy-2′-halogeno, suitably 2′-deoxy-2′-fluoro ribonucleotides; and a morpholino nucleotide.   
     
     
         5 . The method of  claim 1 , wherein the at least one oligonucleotide probe conforms to general formula II, set out below:
   B—[C] n —Y  II
   wherein B is an affinity label that is tethered to oligonucleotide sequence Y via a spacer group C comprising a linear chain of n atoms; the oligonucleotide sequence Y comprising at least 10 nucleotides of which no less than 10% are modified nucleotide analogues; typically at least 25% of the nucleotides are nucleotide analogues; and optionally up to 100% of the nucleotides are nucleotide analogues.   
     
     
         6 . The method of  claim 5 , wherein the spacer group is linked to the 5′ nucleotide of the oligonucleotide probe. 
     
     
         7 . The method of  claim 5 , wherein the nucleotide analogues are selected from 2′ modified ribonucleotide analogues, including 2′-O—R sugar modifications, wherein R is selected from the group consisting of: methyl; ethyl; C 1  to C 5  alkyl; and aryl. 
     
     
         8 . The method of  claim 5 , wherein the nucleotide analogues are selected from 2′ substituted ribonucleotide analogues including 2′-deoxy-2′-halogeno, suitably 2′-deoxy-2′-fluoro ribonucleotides. 
     
     
         9 . The method of  claim 1 , wherein the at least one oligonucleotide probe conforms to general formula III, set out below:
   B—[C] n —P  III
   wherein B is an immuno-tag or a hapten that is tethered to an peptide nucleic acid (PNA) sequence P via a spacer group C comprising a linear chain of n atoms.   
     
     
         10 . The probe of  claim 9 , wherein the spacer group is linked to the N terminal residue of the PNA, P. 
     
     
         11 . The probe of  claim 9 , wherein the spacer group is linked to the C terminal residue of the PNA, P. 
     
     
         12 . The method of  claim 1 , wherein the at least one oligonucleotide probe conforms to general formula IV, set out below:
   B—[C] n -M  IV
   wherein B is an immuno-tag or a hapten that is tethered to a morpholino oligonucleotide sequence M via a spacer group C comprising a linear chain of n atoms.   
     
     
         13 . The probe of  claim 12 , wherein the spacer group is linked to the 5′ nucleotide of the morpholino oligonucleotide sequence M. 
     
     
         14 . The method of  claim 1 , wherein the at least one oligonucleotide probe comprises a group that conforms to general formula V, set out below: 
       
         
           
           
               
               
           
         
         wherein B and B′ comprise an affinity label that is the same or different tethered to respective nucleotides Z and W by spacer groups C and C′ of n atoms in length; and 
         wherein the nucleotides Z and W are separated by an oligonucleotide chain T of p nucleotides in length, where p is between 0 and 40, the nucleotides Z, W and T being selected suitably from a ribonucleotide, a deoxyribonucleotide, a dideoxyribonucleotide and a modified nucleotide analogue, 
         the modified nucleotide analogue being selected from:
 a locked nucleic acid nucleotide (LNA), 
 a 2′ modified ribonucleotide analogue, including 2′-O—R sugar modifications, wherein R is selected from the group consisting of: methyl; ethyl; C 1  to C 5  alkyl; and aryl; and 
 a 2′ substituted ribonucleotide analogue, including 2′-C, and 2′-deoxy-2′-halogeno, suitably 2′-deoxy-2′-fluoro ribonucleotides. 
 
       
     
     
         15 . The method of  claim 14  wherein the at least one oligonucleotide probe comprises the group of general formula III such that nucleotide Z represents the 5′ nucleotide in the oligonucleotide probe. 
     
     
         16 . The method of  claim 1 , wherein the hapten is selected from the group consisting of: biotin or an analogue thereof, such as desthiobiotin; digoxigenin; fluorescein; and dinitrophenol. 
     
     
         17 . The method of  claim 1 , wherein a plurality of oligonucleotide probes are used, and wherein each oligonucleotide probe hybridises to a different portion of the target DNA sequence. 
     
     
         18 . The method of  claim 1 , wherein the target DNA sequence is comprised within one or more of the group consisting of: a telomere; a centromere; euchromatin; heterochromatin; intergenic regions; a gene; a repeat sequence; a heterologously inserted sequence; and an integrated viral genome. 
     
     
         19 . A method of screening for a modulator of epigenetic activity comprising:
 isolating a non-coding nucleic acid that is identified as associating with a specific region of chromatin in the genome of a eukaryotic cell according to the method of  claim 1 ;   contacting the isolated non-coding nucleic acid with one or more compounds from a library of compounds; and   identifying those compound(s) that bind to and modulate the activity of the isolated non-coding nucleic acid as modulators of epigenetic activity.   
     
     
         20 . A method of characterising the biological activity of a non-coding nucleic acid comprising the steps of:
 isolating a non-coding nucleic acid that is identified as associating with a specific region of chromatin in the genome of a eukaryotic cell according to the method of  claim 1 ;   generating an antisense nucleic acid sequence that is complementary to all or a part of the sequence for the a non-coding nucleic acid;   introducing the antisense nucleic acid sequence into a eukaryotic cell so as to deplete the endogenous level of the non-coding nucleic acid in the cell; and   analysing the phenotype of the eukaryotic cell so as to determine the biological activity of the a non-coding nucleic acid.   
     
     
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         25 . A nucleic acid analogue probe, suitable for use in PICh/RICh, as set out in formula I below:
   A—[C] n —X  I
   wherein A includes one or more affinity labels tethered to a nucleotide analogue X by a spacer group C of n atoms in length; A comprises a hapten or an immuno-tag; and wherein the nucleotide analogue X is selected from a peptide nucleic acid (PNA); a 2′ modified ribonucleotide analogue, including 2′-O—R sugar modifications, wherein R is selected from the group consisting of: methyl; ethyl; C 1  to C 5  alkyl; and aryl; and   a 2′ substituted ribonucleotide analogue, including 2′-C and 2′-deoxy-2′-halogeno, suitably 2′-deoxy-2′-fluoro ribonucleotides; and a morpholino nucleotide.   
     
     
         26 . A nucleic acid analogue oligonucleotide probe, suitable for use in PICh/RICh, conforming to general formula II, set out below:
   B—[C] n —Y  II
   wherein B is an affinity label that is tethered to oligonucleotide sequence Y via a spacer group C comprising a linear chain of n atoms; the oligonucleotide sequence Y comprising at least 10 nucleotides of which no less than 10% are nucleotide analogues; typically at least 25% of the nucleotides are nucleotide analogues; and optionally up to 100% of the nucleotides are nucleotide analogues.   
     
     
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